Bottom Line:
However, presence of the residual A. butzleri in the final effluent still deserves more concerns.The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment.Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

Affiliation: State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China.

ABSTRACTThis study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

pone.0125549.g004: Metagenomic analyses of virulence factors (VFs) at six locations along sewage flow in the STP.Sequence number (A) and types (B) of virulence factors were calculated based on alignment of the Illumina shotgun sequences (normalized to 9,000,000 reads for each sample) against MvirDB protein database.

Mentions:
BLAST against MvirDB protein database showed that 716 sequences from SI, 507 sequences from PE, 10 sequences from AS, 18 sequences from SE, 9 sequences from FFE and 6 sequences from FRE were annotated as the known pathogenicity islands, which were assigned to 94, 81, 9, 12, 3 and 2 types, respectively. A total of 208 sequences from SI, 165 sequences from PE, 13 sequences from AS, 15 sequences from SE, 9 sequences from FFE and 4 sequences from FRE were assigned to 31, 23, 5, 9, 5 and 4 types of known virulence proteins, respectively. Similar to pathogens, both pathogenicity islands and virulence proteins in the STP were mainly removed by oxidation ditch (Fig 4), which agrees with the results in absolute terms (S6 Table).

pone.0125549.g004: Metagenomic analyses of virulence factors (VFs) at six locations along sewage flow in the STP.Sequence number (A) and types (B) of virulence factors were calculated based on alignment of the Illumina shotgun sequences (normalized to 9,000,000 reads for each sample) against MvirDB protein database.

Mentions:
BLAST against MvirDB protein database showed that 716 sequences from SI, 507 sequences from PE, 10 sequences from AS, 18 sequences from SE, 9 sequences from FFE and 6 sequences from FRE were annotated as the known pathogenicity islands, which were assigned to 94, 81, 9, 12, 3 and 2 types, respectively. A total of 208 sequences from SI, 165 sequences from PE, 13 sequences from AS, 15 sequences from SE, 9 sequences from FFE and 4 sequences from FRE were assigned to 31, 23, 5, 9, 5 and 4 types of known virulence proteins, respectively. Similar to pathogens, both pathogenicity islands and virulence proteins in the STP were mainly removed by oxidation ditch (Fig 4), which agrees with the results in absolute terms (S6 Table).

Bottom Line:
However, presence of the residual A. butzleri in the final effluent still deserves more concerns.The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment.Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

Affiliation:
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China.

ABSTRACTThis study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.